Genetic Variation of Blast (Pyricularia oryzae Cavara) Resistance in the Longistaminata Chromosome Segment Introgression Lines (LCSILs) and Potential for Breeding Use in Kenya

In Kenya’s rice-growing areas, Basmati varieties have been produced in monoculture since the late 1980s. This has resulted in the breakdown of the resistance (R) gene-mediated response of the local Basmati varieties to blast disease caused by Pyricularia oryzae. To improve blast resistance in Kenyan Basmati varieties, continuous identification of R genes and suitable breeding materials for Basmati are necessary. Longistaminata chromosome segment introgression lines (LCSILs) with the Kernel Basmati genetic background, developed using a rice line called potential low-input adaptable-1 (pLIA-1) derived from a cross between Taichung 65 (T65) (a rice variety in the Japonica Group) and O. longistaminata, are expected to contain useful blast R genes derived from O. longistaminata or T65. In this study, we investigated the genetic variation of blast R genes in LCSILs and their parents by using a new international differential system for designating blast races based on the gene-for-gene theory and molecular characterization using single nucleotide polymorphism (SNP) markers. LCSILs and their parents were classified into three groups—A, B1, and B2—based on reaction patterns to the standard differential blast isolates (SDBIs). Group A, including pLIA-1, showed the highest resistance in all groups, followed by groups B1 and B2. Kernel Basmati in group B1 was considered to possess Pik-p or Pi7(t), Pi19(t), and other unknown R genes. In addition to these R genes, LCSIL 6, 12, 27, 28, and 40, in group A, were determined to possess one of Pish, Piz-t, or both genes that confer resistance to the Kenyan blast races. These lines can be used for efficiently pyramiding blast R genes in the local Basmati varieties.


Introduction
In sub-Saharan Africa, the demand for rice has been increasing in recent decades [1]. In Kenya, rice consumption has been rising with population growth and changes in eating habits [2,3]. Kenya imported 250 million USD of rice in 2020 [1]. Thus, domestic rice production must be expanded along with the increased demand. However, rice productivity in Kenya is suppressed by various biotic and abiotic stresses [4]. Among them, rice blast disease is the major constraint to rice production [5][6][7]. In the Mwea irrigation scheme, producing more than 80% of rice in Kenya, the main variety grown since the late 1980s has been Basmati [3][4][5]. In the Kenyan market, Basmati rice is preferred, and its price is approximately twice as high as that of non-aromatic rice [8]. However, several studies have
of LCSILs and evaluated R genes that were associated with blast resistance. Elite together with the genetic information of QTL(s) for enhancing blast resistance in K Basmati varieties, were obtained based on these results.

Molecular Characteristics of LCSILs and pLIA-1
The molecular characterization of pLIA-1 and LCSILs was carried out using t formation obtained from the GBS analysis. Using 1285 single nucleotide polymorp (SNPs), the parental line, pLIA-1, was observed to have 88.8% of the T65 genome, of the O. longistaminata genome, and 0.2% missing information. The O. longistaminata mosomal segments were identified on chromosomes (chr.) 1, 2, 3, 5, 6, 7, 8, 10, 11, a ( Figure 1). The chromosomal segments from O. longistaminata in the pLIA-1 ranged 0.0-24.5%, with chr. 6 having the highest percentage introgression. Oryza longista chromosomal segments were uniquely identified at the 6.6 Mb region of chr. 2, th Mb region of chr. 3, the 6.4 Mb region of chr. 5, and the 21.6 Mb region of chr. 11 of 1. As for LCSILs, 48 lines were characterized using 1705 high-quality SNPs (Figu The graphical genotype of the LCSILs is presented in Figure 2. Using these inform markers, we identified chromosomal regions that have pLIA-1 segments. The geno LCSILs carried an average of 95.7% Kernel Basmati genome, 3.9% pLIA-1 genom 0.3% heterozygous segments. A total of 159 donor segments were detected in the LC and 128 were evenly distributed in 12 chromosomes. The LCSIL population used study followed the theoretical percentage for a recurrent parent genome (93.8%) BC3 generation.
There were 13 lines of LCSILs in which regions of R genes carried by DVs wer stituted with pLIA-1. The substitution segments in LCSIL 6, 12, 27, and 28 were loca the Pish locus; LCSIL 1 and 5 at the Pit locus; LCSIL 16, 34, and 36 at the Pia locus; As for LCSILs, 48 lines were characterized using 1705 high-quality SNPs ( Figure 2). The graphical genotype of the LCSILs is presented in Figure 2. Using these informative markers, we identified chromosomal regions that have pLIA-1 segments. The genotyped LCSILs carried an average of 95.7% Kernel Basmati genome, 3.9% pLIA-1 genome, and 0.3% heterozygous segments. A total of 159 donor segments were detected in the LCSILs, and 128 were evenly distributed in 12 chromosomes. The LCSIL population used in this study followed the theoretical percentage for a recurrent parent genome (93.8%) at the BC 3 generation. 29 and 48 at the Pik locus; and LCSIL 26, 28, and 40 at the Piz locus. In addition, the stitution segment in LCSIL 8 was located at the pi21(t) locus [39].

Classification of LCSILs Based on the Blast Resistance Phenotyping
LCSILs and their parents showed different reaction patterns to SDBIs (Table Some of the LCSILs scored quite differently to Kernel Basmati, which is their genetic b ground ( Figure 3). The LCSILs' donor line, pLIA-1, showed high or moderate resis to the SDBIs used for testing. In detail, pLIA-1 showed increased resistance to two races, similarly to what was observed in Kernel Basmati, and higher resistance than K Basmati to eight SDBIs. There were 13 lines of LCSILs in which regions of R genes carried by DVs were substituted with pLIA-1. The substitution segments in LCSIL 6, 12, 27, and 28 were located at the Pish locus; LCSIL 1 and 5 at the Pit locus; LCSIL 16,34, and 36 at the Pia locus; LCSIL 29 and 48 at the Pik locus; and LCSIL 26, 28, and 40 at the Piz locus. In addition, the substitution segment in LCSIL 8 was located at the pi21(t) locus [39].

Classification of LCSILs Based on the Blast Resistance Phenotyping
LCSILs and their parents showed different reaction patterns to SDBIs (Table S1). Some of the LCSILs scored quite differently to Kernel Basmati, which is their genetic background ( Figure 3). The LCSILs' donor line, pLIA-1, showed high or moderate resistance to the SDBIs used for testing. In detail, pLIA-1 showed increased resistance to two blast races, similarly to what was observed in Kernel Basmati, and higher resistance than Kernel Basmati to eight SDBIs.
Cluster analysis for the infection scores to the 12 SDBIs showed that LCSILs, Kernel Basmati, and pLIA-1 were classified into two clusters, group A and group B (Figure 4). Group B was further classified into group B1 and group B2. Group A included 13 LCSILs and pLIA-1, group B1 included 24 LCSILs and Kernel Basmati, and group B2 included seven LCSILs.

Classification of LCSILs Based on the Blast Resistance Phenotyping
LCSILs and their parents showed different reaction patterns to SDBIs (Table S1). Some of the LCSILs scored quite differently to Kernel Basmati, which is their genetic background ( Figure 3). The LCSILs' donor line, pLIA-1, showed high or moderate resistance to the SDBIs used for testing. In detail, pLIA-1 showed increased resistance to two blast races, similarly to what was observed in Kernel Basmati, and higher resistance than Kernel Basmati to eight SDBIs.  Plants 2023, 12, x Cluster analysis for the infection scores to the 12 SDBIs showed that LC Basmati, and pLIA-1 were classified into two clusters, group A and group B Group B was further classified into group B1 and group B2. Group A include and pLIA-1, group B1 included 24 LCSILs and Kernel Basmati, and group seven LCSILs. There were differences among the three clusters in infection scores for lates except JPF494 and JPF509 (Table 1). The mean score was lowest in group est in group B2. Group A showed higher resistance than group B1 to seven There were differences among the three clusters in infection scores for all blast isolates except JPF494 and JPF509 (Table 1). The mean score was lowest in group A and highest in group B2. Group A showed higher resistance than group B1 to seven SDBIs and higher resistance than group B2 to six SDBIs. Furthermore, group B1 showed higher resistance to the three SDBIs than group B2.

Evaluation of Blast Resistance Genes
Based on a comparative analysis of the response patterns of the infection scores of the DVs and the tested rice lines to SDBIs, all LCSILs are expected to possess several R genes (Table S1). Table 2 shows the results of the evaluation of blast R genes for each cluster group and the parents of LCSILs. Kernel Basmati was presumed to possess either Pik-p or Pi7(t) and unknown gene(s). Although pLIA-1 was assumed to contain unidentified blast R genes, this differential system could not infer the specific genes because it showed resistance to all 12 SDBIs. T65 was presumed to possess Pish and unknown gene(s). The presumed genotypes of LCSILs showed different trends for each group classified by cluster analysis. Most of the LCSILs classified in group A were presumed to possess one of the Pish, Pib, Pia, and Pii double alleles, one of the Pik double alleles, Piz-t, Pi20(t), and unknown gene(s). The LCSILs classified as group B1 were presumed to possess one of Pik-m, Pik-p or Pi7(t), and unknown gene(s), while group B2 was presumed to contain one of Pik-m, Pik-h, Pik-p, or Pi7(t) and unknown gene(s). However, LCSIL 29 and 48, classified as group B2, were the only LCSILs not presumed to possess Pik double alleles among all LCSILs. Table 2. Evaluation of putative blast resistance genes for each resistance group shown in Figure 4.

Cluster Group /Varieties/Lines Expected Resistance Genes
Indecipherable, Unknown(s)

QTL Mapping of Blast Resistance Loci in LCSILs
Using the SNP and phenotypic data obtained with the 12 SDBIs, QTL analysis for blast resistance was carried out and resulted in the identification of 14 blast resistance QTL (qBR) (LOD > 3.0) in the rice genome ( Figure S1). The qBRs were identified on chr. 1, 2, 3, 4, 7, 8, 11, and 12, wherein phenotypic variance explained (PVE) by each QTL ranged from 27.8% to 69.9%. Among the 14 qBRs, four (qBR1.1, qBAL8, qBR4, and qBR11.2) had a PVE greater than 50.0% and an LOD score > 6.0. The major QTL qBR4 was identified at the 20.5 Mb region of chr. 4 with an LOD score of 11.7 and 69.9% PVE and was observed to be associated with the NIG1 isolate. The qBR11.2, associated with the PHL10 isolate, was identified at the 28.0 Mb region of chr. 11. This QTL had an LOD score of 7.9 and 55.8% PVE. The major QTL qBR1.2, associated with the BEN43 blast isolate, was identified at the 32.5 Mb region of chr. 1 and had an LOD score of 7.2 and 52.5% PVE. Lastly, the major QTL qBR8 was placed at the 211 Kb region of chr. 8 and was observed to be associated with JPF494. This QTL had an LOD score of 7.2 and 50.1% PVE. Several other qBRs were identified in our study with PVE (%) < 50.0%. For example, several qBRs that were associated with the NIG1 blast isolate and mapped to chr. 1 (qBR1.1), 3 (qBR3), and 7 (qBR7) were identified. Each accounted for 31.3% of PVE. On chr. 11, the qBR11.1, which was observed to be associated with the KNY135 blast isolate, was mapped to the 7.3 Mb region. This QTL had an LOD score of 3.7 and accounted for 31.8% PVE. Similarly, qBR11.2, associated with the LAO12 blast isolate, was mapped at 21.4 Mb. This QTL accounted for 37.2% PVE and had an LOD score of 4.5. The qBR12 identified at the 27 Mb region was observed to be associated with BEN43 and accounted for 31.1% PVE.
The summary of the identified qBRs is presented in Table 3. We observed co-localization of resistance in regions of chr. 1 and 11. For example, qBRs associated with PHL12, BEN49, and BEN43 were co-localized in the 32.5 Mb region of chr. 1. Similarly, qBRs associated with LAO12 and PHL10 were co-localized in the 28.0 Mb region of chr. 11.

Detection of Blast Resistance Genotypes of LCSILs and Their Parents
Blast R genes in LCSILs could be assigned using the international differential system. We validated and further narrowed them down by comparing them with the results of genetic analyses. The reaction patterns of most LCSILs to SDBIs were similar to those of Kernel Basmati (Figure 4). The differences between the lines were attributed to the introduction of chromosome segments from the donor parent, pLIA-1.
In the study by Gichuhi et al. [33], an average of one introgression was observed in the LCSILs, whereas in this study, an average of three donor segments per line was observed, likely because a larger number of markers were utilized. The presence of substitutions with chromosomal segments of pLIA-1 in the blast resistance locus region determined using the international differential system matched the locus regions of the Pish, Pia, Pik double alleles, and the Piz double alleles in the ten LCSILs ( R, M, and S indicate resistant, moderate-resistant, and susceptible. LCSIL 6, 12, 27, and 28, presumed to possess Pish, were classified as group A and showed strong or moderate resistance to all blast races except NIG1. The Pish locus region of the donor parent, pLIA-1, was most likely derived from T65 (Figure 1 [38]). T65 has been reported to possess Pish [40,41] and was found to likely contain Pish in this study (Table 2). LCSIL 16,34, and 36, presumed to possess Pia, were classified as group A with strong resistance. The Pia locus region on chr. 11 of pLIA-1 was derived from either T65 or O. longistaminata (Figure 1 [38]). Our results suggested that T65 was not likely to possess Pia, which was consistent with the results of previous studies [40,41]. Therefore, Pia was most likely derived from O. longistaminata.
In LCSIL 29 and 48, the region containing the Pik double alleles was substituted with pLIA-1, and these lines were presumed to be the only LCSILs without either of the Pik double alleles. Thus, Kernel Basmati, the genetic background of LCSILs, was considered to possess one of the Pik double alleles, whereas pLIA-1 did not. Fukuta et al. [7] found that Basmati 217 and Basmati 370 contained one of the Pik double alleles. Furthermore, Kim et al. [42] found that many Basmati varieties, including Basmati 370, possessed Pik, Pik-m, and Pik-p using SNP markers. Therefore, Kernel Basmati was considered to have Pik-p or Pi7(t).
LCSIL 28 and 40, presumed to have Piz-t, were classified as group A with strong blast resistance. The region containing the Piz-t locus on chr. 6 of pLIA-1 was derived from T65 or O. longistaminata (Figure 1 [38]). T65 was presumed not to possess Piz double alleles (Table S1 [40,41]). Furthermore, chr. 6, the locus of Piz-t, had the highest percentage of O. longistaminata substitutions (Figure 1). Therefore, Piz-t, which was presumed to be present in LCSIL 28 and 40 in this study, was likely to be derived from O. longistaminata. In addition to LCSIL 28 and 40, the region containing the Piz locus of LCSIL 26 was also substituted with pLIA-1. However, unlike the other two lines, LCSIL 26 was classified as group B1, which was not in the resistant group. This may be because the expression of the introgressed resistant gene was affected by the length of introgression segments and other resistant genes in the genetic background [43,44].
Analysis of response patterns in the rice blast inoculation test either showed that all LCSILs and Kernel Basmati possess Pi19(t), one of the Pita double alleles, or that the presence of Pi19(t) was masked by other R genes (Table S1). Furthermore, in this study, the region of the Pita double alleles was not substituted in all LCSILs. Therefore, it was reasonable to assume that all LCSILs and Kernel Basmati have Pi19(t). This was supported by the fact that Basmati 217 and Basmati 370 were thought to include Pi20(t), one of the Pita double alleles [7]. Considering the results of the genetic analysis and the evaluation of R genes based on the response patterns in the rice blast inoculation test, the parental variety Kernel Basmati was considered to possess Pi19(t) and unknown genes in addition to Pik-p or Pi7(t), while pLIA-1 contained at least Pish, Pia, and Piz-t but no Pik double alleles. Fukuta et al. [7] reported that Basmati 217 and Basmati 370 were classified as the most resistant group of the varieties grown in Kenya to the Japanese, Philippine, and Kenyan blast races, and they were presumed to possess Pi20(t), one of the Pik double alleles or Pi3, and unknown gene(s). Therefore, the parental variety Kernel Basmati was considered to have a similar level of resistance to Basmati 217 and Basmati 370 among the varieties grown in Kenya. However, in areas where Basmati varieties are widely grown, they are generally known to be susceptible to blast [45][46][47]. In Kenya, Basmati varieties have been reported to be severely damaged by blast [5][6][7]. In these areas, blast races that can infect Basmati varieties are dominant because Basmati varieties have been grown for many years [7,48].
In this study, LCSILs and their parents were presumed to contain Pik-p or Pi7(t), Pish, Pia, Piz-t, Pi19(t), and unknown R genes. Among these, Piz-t has been reported to show resistance to many blast races when introduced into susceptible lines [49][50][51]. Piz-t showed resistance in more than three Kenya regions and is considered a stable R gene. Pish, which showed resistance in some regions, was also considered a useful gene with high stability. On the other hand, Pik-p, Pi7(t), and Pi19(t) are at increased risk of resistance breakdown and may become susceptible to blast within a few years. LCSIL 28, which showed good resistance in this study, was presumed to possess Pish, Pik-p or Pi7(t), Piz-t, and Pi19(t) ( Table 4), as well as R genes other than the 23 genes in the DVs. In addition, LCSIL 26 and 40, presumed to harbor Piz-t and LCSIL 6, 12, and 27, presumed to possess Pish, would be useful breeding material for blast-resistant Basmati varieties for Kenya. Furthermore, LCSIL 8,19,36,41, and 50, classified as Group A, showed good resistance to many SDBIs. However, their reaction patterns differed from those of the DVs, suggesting that their blast resistance was conferred by genes other than the 23 R genes introduced in the DVs. Among them, LCSIL 8, in which pLIA-1 was introduced into the pi21(t) locus region on chr. 4 [39], may possess pi21(t) from T65 (Figure 1 [38]).
This study detected 14 rice blast resistance QTLs, including qBR1.2, qBR1.3, and qBR1.4 for resistance to BEN43, PHL12, and BAN491, and qBR11.3 and qBR11.4 for resistance to PHL10 and LAO12, which were identified in the regions containing Pish and Pik double alleles, respectively. Furthermore, qBR11.1 for resistance to KNY135 was identified near the Pia locus. In addition, qBR4 for resistance to NIG1 was identified near pi21(t) [39], although it is not included in the R genes of the DVs. The QTLs qBR3 and qBR7 convey resistance to NIG1, and qBR12 conveys resistance to BEN43. LCSILs 14 and 50 substituted with pLIA-1 in these regions showed strong resistance to the respective blast races. Thus, qBR3, qBR7, and qBR12 likely contain new R genes that have not yet been reported. Since pLIA-1, the donor parent of the LCSILs, contains chromosome segments of O. longistaminata (Figure 1 [38]), some of the novel blast R genes may be derived from O. longistaminata. Further research is needed to identify these novel blast-resistant genes.

Potential for Breeding Use of LCSILs
Blast resistance in Basmati has not been reported as much as in japonica varieties. Moreover, the breeding of blast-resistant Basmati varieties is not as advanced as with japonica varieties. One of the major challenges in breeding Basmati varieties is hybrid sterility. Hybrid sterility is known to occur in crosses between japonica and indica varieties [52,53], and Basmati varieties have been reported to be hybrid sterile with japonica and indica varieties [10].
To date, some blast-resistant lines have been produced by introducing Pi9(t) into Ranbir Basmati with a genetic background of Basmati 370 [47]; Pi2 and Pi54 into Pusa Basmati 1121 and Pusa Basmati 6 [54]; and Pi2 (=Piz-5), Pi9(t), Pi1, Pi54, Pita, Pib, and Pi5(t) into Pusa Basmati 1 [46]. However, these lines are not available in Kenya. Few useful materials are available for breeding for blast resistance in Basmati. Using LCSIL 6, 12, 27, 28, and 40, which have Kernel Basmati genetic backgrounds, as breeding material for crosses, it will be possible to develop blast-resistant Basmati for Kenya carrying Piz-t and Pish without causing hybrid sterility. LCSILs other than these selected lines can also introduce other R genes such as Pia, Pik double alleles, etc. In addition, LCSILs were found to possess several unknown R gene(s), some of which were likely derived from the African wild rice O. longistaminata. Further research is needed to investigate the potential breeding use of these unknown R gene(s).

Plant Materials
The breeding population used in the study is comprised of LCSILs, which have the genetic background of Kernel Basmati, and part of the chromosome has been replaced by a pLIA-1 chromosome [33]. pLIA-1 is a rice line derived from an interspecific cross between the wild rice species, O. longistaminata, and a rice cultivar, T65 [32]. It was initially described to have a large biomass even when grown under non-fertilized conditions [33,38,55]. On the other hand, Kernel Basmati is an aromatic rice variety with excellent grain quality. In summary, F 1 hybrids were backcrossed to the recurrent parent, Kernel Basmati, to obtain BC 1 F 1 plants. A total of 22 plants were selected, and successive backcrossing was conducted to obtain BC 2 F 1 plants. In the BC 3 F 1 generation, a total of 55 plants were selected and selfed. A total of 50 lines were selected at the BC 3 F 5 , BC 3 F 6 , and BC 3 F 7 generations ( Figure 5).
Plants 2023, 12, x wild rice O. longistaminata. Further research is needed to investigate the potential b use of these unknown R gene(s).

Plant Materials
The breeding population used in the study is comprised of LCSILs, which h genetic background of Kernel Basmati, and part of the chromosome has been rep a pLIA-1 chromosome [33]. pLIA-1 is a rice line derived from an interspecific c tween the wild rice species, O. longistaminata, and a rice cultivar, T65 [32]. It was described to have a large biomass even when grown under non-fertilized co [33,38,55]. On the other hand, Kernel Basmati is an aromatic rice variety with e grain quality. In summary, F1 hybrids were backcrossed to the recurrent parent Basmati, to obtain BC1F1 plants. A total of 22 plants were selected, and successi crossing was conducted to obtain BC2F1 plants. In the BC3F1 generation, a total of 5 were selected and selfed. A total of 50 lines were selected at the BC3F5, BC3F6, an generations ( Figure 5).

Genotyping
Plant materials, including 48 lines of LCSILs, parental lines of LCSILs, Ker mati, and pLIA-1 were grown in the paddy field at the Higashiyama campus of University, Nagoya, Japan, in 2020. They were sown in early June and transplante

Genotyping
Plant materials, including 48 lines of LCSILs, parental lines of LCSILs, Kernel Basmati, and pLIA-1 were grown in the paddy field at the Higashiyama campus of Nagoya University, Nagoya, Japan, in 2020. They were sown in early June and transplanted in late June. The leaf materials from three plants of each line were harvested in late November and dried at 56 • C for 24 h.
The GBS library for the LCSILs was prepared following the protocol of Poland et al. [56] and Furuta et al. [57]. In summary, genomic DNA (200 ng) of plant material was extracted using the cetyltrimethylammonium bromide (CTAB) method [58]. The DNA quality and quantity were analyzed using a 1% agarose gel and Quantus TM Fluorometer (Promega, Madison, WI, USA). The DNA concentration of each sample was then adjusted to 20 ng/uL and double-digested using a combination of restriction enzymes KpnI-MspI. The doubledigested DNA samples were ligated with unique barcode adapters and pooled into a single tube. The library sequencing was carried out using Illumina Miseq (Illumina, Inc., San Diego, CA, USA).
The informatics processing of the GBS reads was conducted using the TASSEL-GBS 5.0 pipeline [59]. In summary, the obtained 64 bp long sequence tags were aligned to the IRGSP V1.0 O. sativa Nipponbare reference genome sequence [60] using the Burrows-Wheeler Aligner (BWA) [61]. The SNPs were called by filtering the minimum allele frequency (mnMAF) to 0.02 and the minimum locus coverage (mnLCov) to 0.8. The obtained reads were further filtered based on polymorphism between parental alleles using an awk script written by the authors, and the remaining low-quality markers were manually removed.

Phenotyping for Blast Resistance
Blast resistance of 45 lines of LCSILs, Kernel Basmati, pLIA-1 and T65 were evaluated with 28 DVs, including 25 DVs that included monogenic lines and NILs, by inoculation tests targeting 23 R genes [19].  [63]. LTH and US-2 were evaluated as the susceptible control variety.
Stock isolates were cultured on an oatmeal agar medium and incubated at around 25 • C for 12 to 13 days. The culture plate was scraped using a toothbrush and exposed to fluorescent light for 4 to 5 days to induce heavy sporulation. Using a paintbrush, conidia were dislodged from the plate into 10 to 20 mL of sterilized distilled water containing 0.01% of tween 20. Spore suspensions were filtered, and spore concentration was adjusted to 1 × 10 5 conidia per mL using a hemacytometer [68]. Plants were grown in a greenhouse for 2 weeks (approximately 4-to 5-leaf stage) and inoculated by spraying with a spore suspension. After inoculation, the seedlings were incubated at 25 • C at a relative humidity of 70 to 80% for 20 h and then transferred to a greenhouse at a relative humidity of approximately 60% and a temperature of around 25 • C.
The infection score of each isolate was evaluated based on the 6-rating system 7 days after inoculation [69]. The experiment was performed twice. For evaluating the R genes, a score of 0 to 2 was categorized as resistant (R), 2.5 to 3 as moderate resistance (M), and 3.5 to 5 as susceptible (S). By comparing the DVs' reaction patterns to the SDBIs, we evaluated the resistance genes for LCSILs and parental lines and varieties.

QTL Analysis for Blast Resistance
The QTL analysis was performed by CSL function and single-marker analysis (SMA) implemented in the QTL IciMapping ver. 4.1 [70]. The threshold LOD value was determined by a permutation test involving 1000 runs at a significance level of p = 0.05, and the QTL in a particular genomic region with LOD values larger than this threshold for each trait were used.

Statistical Analysis
Cluster analysis using Ward's hierarchical method was performed for the classification of introgression lines to SDBIs. The infection scores of all lines were analyzed using the Shapiro-Wilk normality test. Since these replicated data did not show a normal distribution, the nonparametric Steel-Dwass test was performed following a Kruskal-Wallis test to evaluate the differences between the groups classified by cluster analysis. All statistical analyses were performed using the averages of two inoculation experiments by JMP ver. 16 (SAS Institute, Inc., Cary, NC, USA).

Conclusions
To control blast in Kenya without excessive reliance on pesticides, it will be necessary to develop new varieties carrying multiple blast R genes, or to grow multiple varieties with different R genes, including multilines (a mixture of near-isogenic lines with varying sources of resistance). However, few reports of such measures being applied in developing countries are available. Furthermore, relevant studies using Basmati varieties are extremely limited. In this study, blast resistance phenotypes and genotypes of LCSILs were evaluated by the international differential system and genetic analysis, and lines showing high resistance to the Kenyan blast races were selected. The selected lines are LCSIL 6, 12, 27, 28, and 40, which can be used for efficiently pyramiding blast R genes in the local Basmati varieties. They will also be useful in developing multilines with different blast R genes. Thus, the findings of this study can provide a significant contribution to overcoming rice blast disease in Kenya.

Supplementary Materials:
The following are available online at: https://www.mdpi.com/article/ 10.3390/plants12040863/s1. Table S1: Response patterns of LCSILs, Kernel Basmati, pLIA-1, T65, and international differential varieties (DVs) to 12 standard differential blast isolates (SDBIs). Figure   Data Availability Statement: Data generated in this study is available upon request to the corresponding author.